Since the discovery of RNA interference, it has been clarified that various types of small RNAs are deeply involved in vital activities and diseases caused by abnormalities thereof, such as cancers. Usually, a small RNA is analyzed by being converted to a cDNA with an enzyme, so-called reverse transcriptase, and then measured by a molecular biological method. Such a method has the disadvantages that analysis of chemical modification, which is important for expression of function, and quantitative measurement of the small RNA are impossible.
In known nano-LC-ESI-MS methods having the above-described restrictions, since a hydrophilic nucleotide having (−) charge is eluted from an LC column during a separation process under a low organic solvent concentration, the surface tension of the separate solution is increased. Therefore, the efficiency of ionization in a nano-electrospray ionization technique is decreased, resulting in a problem of a significant decrease in analysis sensitivity. That is, in the nano-electrospray ionization technique in which an LC separation solution having a low organic solvent concentration is used, droplets are generated at the tip of a needle for spraying the separation solution and cause clogging, which makes the spraying intermittent or discontinuous and also the analysis itself impossible.
Against these problems regarding analysis in the micro order (i.e., a flow rate of a micro-litter (μL/min) level), methods have been developed for preventing the formation of droplets at the needle tip, wherein a needle having a double-tube structure in which a “sheath tube” parallel surrounds a needle for spraying separation solution is used. The sheath tube supplies an organic solvent (sheath solution) to the LC separation solution to be sprayed (Journal of Chromatography A, 870, (2000), 413-424).
Though the above-mentioned technology is an effective method for the analysis at the micro-level, the technology has not reached a level for being applied to analysis at the nano-level (i.e., a flow rate at a nano-litter (nL/min) level). This is because that it is technically difficult to form a double-tube structure including a micro sheath tube for suitably supplying a sheath solution to a micro needle having an inner diameter of about 75 to 200 μm used in the nano-LC-ESI-MS method.